Yeast enolase has an absolute requirement for magnesium for activity, though some other divalent metals provide lower levels of activity. Some metals such as calcium permit no activity. There are three classes of metal binding sites on this enzyme. Conformational metal, catalytic and inhibitory metal can be bound, depending on conditions. The proposed research is to examine the effects of each of the classes of metals, and the effects of activating and nonactivating metals on the structure and environment of the substrate and competitive inhibitors, including two which appear to undergo reversible partial reactions if activating metals are present. The techniques employed are 31P- nuclear magnetic resonance, circular dichroism and ultraviolet resonance Raman spectroscopy and stopped-flow studies. Another approach is to study the environments of the different classes of metals themselves, both activating and nonactivating metals, and the effects of substrate and inhibitors on these environments. The techniques used are circular dichroism, electron paramagnetic resonance and absorption spectroscopy, fluorescence lifetime and energy transfer measurements. The objectives of these proposed experiments are to use yeast enolase as a model system to determine the mechanism of action of each class of metals, and the origin of the specificity for some metals for activity.